The long-term goal of this project is to assess potential harmful effects on humans of Roxarsone and related benzenearsonates in drinking water. These compounds are routine feed additives in poultry and swine, because of their antimicrobial and growth-promoting qualities. Concentrated animal farming operations (CAFOs) have burgeoned in this country since the late 1980s. EPA, USGS and public health groups place major blame on farming practices for the declining water quality in the United States. Annually land and waterways receive over 2 million pounds of Roxarsone and its metabolites, and unlisted amounts of the additive Arsanilate and its metabolites in the tons of manure from swine CAFOs. This laboratory has been concerned with human impact of the organoarsenical feed additives, as monitored in vitro with human enzymes and with a human intestinal cell line, Caco-2. Recent work has shown that Roxarsone and its metabolites permeate Caco-2 cells at the [unreadable]M levels where their proliferative effects are observed. Specific aim 1 is to characterize the rate and determine the mechanism of permeation of Caco-2 cells by AHBA and related compounds. Use of Transwell devices for Caco-2 growth and dosing, as well as the laboratory's HPLC/MS capability, should expedite achieving this aim. Absorption from apical to basolateral side in Caco-2s in Transwells mimics absorption across the intestinal wall into the circulatory system. Specific aim 2 is to test the effects of benzenearsonates on cultured human liver HepG2 cells and on cultured hepatocytes. Ingested substances absorbed into the circulation travel next to the liver, a rich source of most enzymes. Benzenearsonate uptake will be measured in these cells by quantifying disappearance from media or appearance in the cells. Proliferation and/or toxicity will be determined by the MTT assay. Specific aim 3 is to observe effects of [unreadable]M level dosing of Arsanilate on synchronous Caco-2 cells. Arsanilate permeation and proliferative effects will be measured in synchronous cultures as described in specific aims 1 and 2. Specific aim 4 is to elucidate the mechanism of AHBA bioactivation. Bioactivation of the metabolite AHBA appears to be favored over detoxification reactions. Human cytochrome P450 enzymes react with AHBA to form unidentified products. These products can produce harmful DNA adducts identifiable by LC/MS, HPLC, UV/Vis, and MS instrumentation are used to separate, quantify, and identify products.